Bianco Mariachiara, Calvano Cosima D, Ventura Giovanni, Losito Ilario, Cataldi Tommaso R I
Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Bari, Italy.
Centro Interdipartimentale di Ricerca SMART, Università degli Studi di Bari Aldo Moro, Bari, Italy.
Methods Mol Biol. 2024;2820:67-88. doi: 10.1007/978-1-0716-3910-8_8.
Two protocols of protein extraction from Arthrospira platensis (spirulina) microalgae to study their proteome by mass spectrometry (MS) are here presented. The first is based on an aqueous buffer solution of Tris-HCl and the second on cold acetone. The identification of proteins was carried out by a bottom-up approach, which involves enzymatic digestion of extracted proteins followed by either matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) MS or liquid chromatography (LC) coupled with electrospray ionization (ESI) and Fourier-transform tandem MS. While MALDI-TOF MS allowed for a fast peptide mass fingerprinting (PMF) check yet identifying less than 20 proteins in the extracted samples, the data-dependent acquisitions (DDA) mode of reversed-phase (RP) LC-ESI tandem FTMS/MS separations allowed us to recognize more than one hundred proteins by searching into dedicated spectral libraries. The application of MALDI-TOF MS analysis was found, however, of great support for preliminary investigations of cyanobacteria samples before proceeding with the RPLC-ESI-MS/MS DDA investigation, which definitively allows for a qualitative proteome analysis also of minor spirulina proteins in processed foodstuffs. Although the protein content in spirulina can be influenced by cultivation and environmental conditions, e.g., light intensity, climate, and water/air quality, here the qualitative chemical profiles of the examined samples were characterized by similar composition in high-quality proteins as phycocyanins and phycoerythrins.
本文介绍了两种从钝顶螺旋藻（螺旋藻）微藻中提取蛋白质的方法，用于通过质谱（MS）研究其蛋白质组。第一种方法基于Tris-HCl水溶液缓冲液，第二种基于冷丙酮。蛋白质鉴定采用自下而上的方法，该方法包括对提取的蛋白质进行酶解，然后进行基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱分析，或液相色谱（LC）与电喷雾电离（ESI）和傅里叶变换串联质谱联用分析。虽然MALDI-TOF质谱分析能够快速进行肽质量指纹图谱（PMF）检查，但在提取的样品中鉴定出的蛋白质不到20种，而反相（RP）LC-ESI串联FTMS/MS分离的数据依赖采集（DDA）模式使我们能够通过在专用光谱库中搜索识别出100多种蛋白质。然而，发现MALDI-TOF质谱分析在进行RPLC-ESI-MS/MS DDA研究之前，对蓝藻样品的初步研究有很大帮助，RPLC-ESI-MS/MS DDA研究最终也能够对加工食品中微量螺旋藻蛋白质进行定性蛋白质组分析。尽管螺旋藻中的蛋白质含量会受到培养和环境条件的影响，例如光照强度、气候以及水/空气质量，但此处所检测样品的定性化学谱特征是含有类似组成的高质量蛋白质，如藻蓝蛋白和藻红蛋白。
